Experimental Study On Human Umbilical Cord Blood Mesenchymal Stem Cells In Combination With β-tricalcium Phosphate For Bone Tissue Engineering

Objective: To culture in vitro hUCB-MSCs with two modified methods, identify its biological characteristics, observe its biocompatibility withβ-TCP, and to investigate the support effect of hCS on the culture and expansion of hUCB-MSCs in vitro.Methods: Full-term hUCB samples were obtained with mother's consent(n=28). Mononuclear cells were separated from hUCB by density gradient centrifugation, then seeded inα-MEM supplemented with 10% FBS. The medium was half changed after 5～7 days'primary culture and then totally changed at a 3～4 days'interval. When the adherence of MNCs happened, the subsequent procedures were taken according to modified methods. The morphology was observed under microscope per day. Flow cytometry was used to examine the immunophenotype. hUCB-MSCs were transfered into an osteogenic, adipogenic and chondrogenic medium respectively,and cytochemical staining was carried out subsequently; combined withβ-TCP, whose growth status was observed under SEM; and cultured inα-MEM supplemented with 10% hCS, whose status of growth, expansion and differentiation was studied.Results: 1) Adherent fibroblast-like cells obtained from 13 of 28 hUCB samples were passaged steadily(Group I, 4/10; Group II, 9/18) with the total success rate of 46.4%. There were two types of adherent cells after 5 to 7 days'primary culture, MLCs and OLCs.MLCs colonies were found after 5 weeks'primary culture,morphologically similar to BM-MSCs,which could be passaged steadily to 22 passages without significant differences. 2) hUCB-MSCs were all strongly positive for MSC-related antigens such as CD29 and CD105. 3) Incubation of hUCB-MSCs under special conditions resulted in a dramatic increase in osteogenic,adipogenic and chondrogenic activity as assessed by corresponding staining. 4) hUCB-MSCs grew well on and interior porousβ-TCP. 5)hUCB-MSCs kept high growth, steady expansion, and osteogenic and adipogenic differentiation after expanded for 5 passages inα-MEM supplemented with 10% hCS.Conclusions: hUCB does contain MSCs. Similar to those from other sources, these MSCs, which could be expanded easily, passaged steadily and differentiated dramatically into osteoblast, adipocyte and chondrocyte, could be cultured more successfully by modified culture methods.Excellent biocompatibility was showed by combination of hUCB-MSCs andβ-TCP. Support of hCS on the culture and expansion in vitro of hUCB-MSCs was showed by initial study. hUCB-MSCs might serve as an alternative seed cell in future bone tissue engineering.